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Structural characterization of the SAC domain of the Par-4 protein by NMR spectroscopy : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Physics at Massey University, Turitea, Palmerston North, New Zealand

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Abstract

Prostate Apoptosis Response-4 protein (Par-4): Par-4 gene, first identified in prostate
cancer cells undergoing apoptosis, encodes a pro-apoptotic protein. Par-4 selectively
induces apoptosis in cancer cells causing regression of tumors in animal models.
Par-4 induces apoptosis in androgen independent prostate cancer cells and Rastransformed
cells but not in androgen-dependent normal cells. Apoptosis induction by
the Par-4 involves a complex mechanism that requires activation of the Fas death
receptor signalling pathway and coparallel inhibition of NF-kB transcription activity.
Par-4 expression is elevated in various neurodegenerative diseases such as
Alzheimer's, Parkinson's, Huntington's diseases and stroke.
Rat Par-4 MW 36kDa, full length aa332 protein that shows high homology to human
Par-4. Par-4 has two putative nuclear localization sequences NLS1 (aa20-25) and
NLS2 (aa 137-153) localized to the N-terminal half of the molecule. SAC domain:
Selectivity for apoptosis induction in cancer cells 59-aa (aa137-195) is necessary for
apoptosis. Leucine zipper(LZ) domain of 41-aa (aa 292-332) at C terminus which
binds to zinc finger domain of aPKC isoforms, Wilms tumor(WT1) protein, p62 and
Dlk. Par-4 Δleucine zip aa1-265, zipperless domain.
Different types of expression vectors have been tried for expression of the Par-4 SAC
domain in E. coli cells. The first expression vector was H-MBP-3C was tried with
E.coli rosetta cells. The fusion protein expressed with this vector was not stable. The
Par-4 SAC domain protein gene sequence was cloned in different trial vectors. In the
beginning we decided to use a vector without any fusion tag, the pPROEX-HTb
which shows low level of expression in E.coli BL21 (DE3) CP cells. The pETTEV
expression vector with a thioredoxin tag shows good expression level and fusion
protein stability. The protein sample after purification was characterized by proton
NMR spectroscopy, CD and DLS which does not show any presence of protein
folding. pH titration by using NMR spectroscopy to simultaneously observe the
protonation state of different ionizing functional groups in peptides and proteins with
both acidic and basic condition was also done. There was no evidence of any protein
folding in the acidic as well as basic pH range. This suggests that protein is natively
unfolded.